Bacterial-derived component removal material

ABSTRACT

A material having affinity for LTA and/or PrA and/or aHL and/or proteinase is prepared by introducing functional groups capable of hydrogen bond formation into a substrate. Using this material, LTA and/or PrA and/or aHL and/or proteinase in blood, plasma or other solution of high protein concentration can be rapidly detoxified or removed. Articles for LTA and/or PrA and/or aHL and/or proteinase removal of detoxification using this material are described, in particular a body fluid purification column for LTA and/or PrA and/or aHL and/or proteinase removal and an LTA and/or PrA and/or aHL and/or proteinase-adsorptive dressing.

TECHNICAL FIELD

The present invention relates to a material having selective affinityfor compounds produced by prokaryotic micro-organisms, namelygram-negative bacteria and gram-positive bacteria. From among thecompounds produced by bacteria, it relates to a material which removesor results in a loss of toxic activity (detoxification) of lipoteichoicacid (hereinafter abbreviated to LTA), protein A (hereinafterabbreviated to PrA), α-hemolysin (abbreviated to aHL) or proteinase. Inparticular, since it binds to the LTA and/or PrA and/or aHL and/orproteinase occurring in blood and other such solutions of high proteinconcentration, it is ideally used either as a medicinal agent whichbrings about a loss of the toxic activity (detoxification) of LTA and/orPrA and/or aHL and/or proteinase, or as a purification column ordressing which removes LTA and/or PrA and/or aHL and/or proteinase.

TECHNICAL BACKGROUND

In recent years, as a result of a variety of studies, LTA has come to beregarded as one of the causal substances of gram-positive bacterialsepsis. For example, it has been reported that it induces expression ofnitric oxide synthase in cultured vascular smooth muscle cells (J.Cardiovasc. Pharmacol., 20, S145-S147 (1992)), that as a result ofintravenous infusion in rats there occurs a reduction in blood pressureand a reduction in the pressor reaction due to noradrenaline (Br. J.Pharmacol, 144, 1317-1323 (1995)), that as a result of intrathoracicadministration to animals, neutrophil infiltration into the thoraciccavity is observed and pleurisy occurs (JP-A-9-163896) and, furthermore,that if LTA and peptidoglycan are jointly administered to rats, shockand internal organ disturbances occur (J. Exp Med., 188 (2), 305-315(1998)). By way of contrast, it has been reported that nitric oxideproduction is inhibited by anti-mouse CD14 antibodies (Biochem BiophysRes Commun, 233 (2), 375-379 (1997)) or by N(omega)-nitro-L-argininemethyl ester (Infect Immun, 65 (6), 2074-2079 (1997)), and that plateletactivating factor antagonist inhibits shock deaths resulting from LTAadministration to mice (JP-A-9-208493). Furthermore, the effects ofaminoguanidine and dexamethasone (Br J Pharmacol. 119 (7), 1411-1421(1996)) have also been investigated. However, these all targetsubstances produced in the latter part of the inflammatory responseinitiated by LTA and there are no reports of drugs which target the LTAitself. Now, regarding materials for medical use, there is a report(ASAIO J, 44 (1), 48-53 (1998) that fibre-immobilized polymyxin-B bringsabout a 20% reduction in the TNF-α production due to a Staphylococcusaureus culture supernatant (diluted with 10% human plasma-containingmedium). However, various toxins are contained in the culturesupernatant and LTA removal has not been confirmed. Moreover, thepercentage removal was also low, at 20%. Thus, there have not been knownhitherto materials which have a high affinity for LTA in high proteinsolutions such as plasma.

In addition, it is known that, as a result of the agglomeration broughtabout by PrA binding to immunoglobulin G (hereinafter abbreviated toIgG), which is the main protein of the human immune system, deactivationof the activity thereof is brought about. As a result of the IgGdeactivation and a lowering of the immune capability, bacteria readilyinvade the body and sepsis is aggravated. In the same way, aHL is aprotein which harms cells by bringing about the formation of pores inthe cell membranes of human cells (particularly blood corpuscles) and,by destroying cells, there is created a situation where infectionreadily occurs and sepsis is aggravated. No materials are known whichhave a characteristic affinity for such toxins, PrA and aHL.

OBJECTIVE OF THE INVENTION

The present invention relates to a material which resolves theseprior-art problems and also has further novel functions, and itsobjective is to provide a material which can rapidly bring about a lossof toxic activity (detoxification) of, or can remove, the LTA and/or PrAand/or aHL and/or proteinase in blood or other such solutions of highprotein concentration.

Specifically, the material of the present invention has a high affinityfor bacterially-derived components and binds LTA and/or PrA and/or aHLand/or proteinase present in blood, plasma and other such body fluids,or in pharmaceutical preparations and, it this way, it is possible todetoxify the activities of these toxins and to treat and prevent sepsisand infectious diseases. Moreover, where this material iswater-insoluble, there can then be provided a material which adsorbs LTAand/or PrA and/or aHL and/or proteinase present in blood, plasma andother such body fluids, or in drugs, by binding such toxins, and byemploying such a material there can be provided a blood purificationcolumn or a wound dressing for the treatment or prevention of sepsis orinfectious disease.

DISCLOSURE OF THE INVENTION

The present invention has the following constitution for resolving theproblems described above.

(1) A bacterially-derived component detoxification or removal materialwhich is characterized in that it has at least one functional groupcapable of hydrogen bond formation and detoxifies or removes at leastone of the bacterially-derived components selected from lipoteichoicacid, protein A, α hemolysin proteinase and endotoxin.

(2) A bacterially-derived component detoxification or removal materialwhich is characterized in that it has a functional group capable ofhydrogen bond formation and a hydrophobic group and/or ether bond, andit detoxifies or removes at least one of the bacterially-derivedcomponents selected from lipoteichoic acid, protein A, α hemolysin,proteinase and endotoxin.

(3) A material for sepsis treatment where a material according to (1)and (2) is employed.

(4) A wound dressing employing a material according to (1) and (2).

(5) A method for the removal or detoxification of lipoteichoic acidand/or protein A and/or a hemolysin and/or proteinase in liquids, usinga material according to (1) and (2).

OPTIMUM FORM FOR PRACTICING THE INVENTION

In the present invention, LTA refers to a substance from which the cellmembrane and cell wall of gram-positive bacteria such as bacteria of thegenera Streptococcus, Micrococcus, Lactobacillus, Staphylococcus,Bacillus and Enterococcus are composed (“Ika Saikingaku [MedicalBacteriology]”, Ed. by Masanosuke Yoshikawa, Published by Nankodo).Furthermore, PrA and aHL are proteins produced by Staphylococcus aureus.Again, in the present invention, proteinase refers tobacterially-derived proteinase which cleaves partial sequences fromprotein precursors, producing active proteins. These bacterially-derivedcomponents are toxins which are highly likely to be involved in theaggravation of infectious diseases, in particular sepsis.

In addition, it is suspected that there is a relationship betweenstreptolysin, coagulase, enterohemorrhagic toxin, pseudomonas exotoxinA, cholera toxin, botulinus toxin, verotoxin, leukocidin, superantigen,endotoxin and the like, and the aggravation of infectious diseases, inparticular, sepsis. Of these, superantigen and endotoxin are highlytoxic and the detoxification or removal of these toxins is desirable atthe same time as that of LTA, PrA and aHL.

In the present invention, there are no particular restrictions on thefunctional groups capable of hydrogen bond formation, and examples arethe urea bond, thiourea bond, urethane bond, amide group, amino group,hydroxyl group, carboxyl group, aldehyde group, mercapto group andguanidino group, but possession of at least one urea bond, thioureabond, amide bond, amino group or hydroxyl group is preferred. There areno particular restrictions on the structure adjoining the group capableof forming a hydrogen bond, and there can be employed aliphaticcompounds such as propane, hexane, octane and dodecane, or alicycliccompounds such as cyclohexane and cyclopentane but, taking intoconsideration their high affinity, aromatic compounds such as benzene,naphthalene or anthracene are more preferably used. Derivatives such asbromoheptane, chlorocyclohexane, methylbenzene, chlorobenzene,nitrobenzene, diphenylmethane and chloronaphthalene are also suitablyemployed. Again, it is further preferred that there be at least twogroups capable of hydrogen bond formation and, in particular, there ispreferably employed a structure which also possesses a functional groupcapable of forming a hydrogen bond, such as an amino group, hydroxylgroup or carboxyl group, as the structure adjoining the urea bond,thiourea bond or amide bond. As examples of compounds with an aminogroup, there are aminohexane, monomethylaminohexane,dimethylaminohexane, aminooctane, aminododecane, amino-diphenylmethane,1-(3-aminopropyl)imidazole, 3-amino-1-propene, aminopyridine,aminobenzenesulphonic acid, tris(2-aminoethyl)amine and the like, morepreferably diaminoethane, diethylenetriamine, triethylenetetramine,tetraethylenepentamine, dipropylenetriamine, polyethyleneimine,N′-methyl-2,2′-diaminodiethylamine, N-acetylethylenediamine,1,2-bis(2-aminoethoxyethane) and other such compounds with a pluralityof amino groups (sometimes referred to as polyamines). Again, asexamples of compounds with a hydroxyl group, there can be usedhydroxypropane, 2-ethanolamine, 1,3-diamino-2-hydroxypropane,hydroxybutanone, hydroxybutyric acid, hydroxypyridine and the like,glucides such as monosaccharides, oligosaccharides and polysaccharidessuch as lucose, glucosamine, galactosamine, maltose, cellobiose,sucrose, agarose, cellulose, chitin, chitosan and the like, andderivatives of these. Furthermore, as examples of structures with acarboxyl group, there can be used β-alanine, n-caproic acid, isobutyricacid, γ-amino-β-hydroxybutyric acid and the like. Most preferably, therecan be used as the material of the present invention a compound both anaromatic group and a compound capable of forming a hydrogen bond asstructures adjoining the urea, thiourea or amide groups.

As a functional group other than a group capable of forming a hydrogenbond, it is preferred that there be present a hydrophobic group or etherbond. Hydrocarbons with at least 4 carbons and aromatic rings arepreferred as the hydrophobic group, and they are effective withoutdistinction in terms of being linear, branched, cyclic, saturated orunsaturated. Specific examples are the n-butyl group, sec-butyl group,tert-butyl group, isobutyl group, n-hexyl group, n-octyl group,n-dodecyl group, cyclohexyl group, benzyl group, 3,3-diphenylpropylgroup n-butylamino group, n-hexylamino group, n-dodecylamino group,n-hexadecylamino group and the like.

As the hydrophobic group, it is preferred that it have log P value atleast 0.7 (P=partition coefficient in an octanol/water system). It ispossible to refer to the published values of a number of known compounds(see Albert Leo, Corwin Hansh and David Elkins, Partition coefficientsand their uses, Chemical Reviews, vol.71, No.6 p525-616 (1971)).

As examples of structures containing an ether bond or ether bonds, thereare straight chain ethers such as the ethoxyethyl group and methoxyethylgroup, cyclic ethers such as crown ethers, and the ethers contained inglucides such as cellulose, agarose and the like.

Furthermore, it is also possible to use polyureas, polythioureas andpolyamides, which contain a plurality of urea bonds, thiourea bonds andamide bonds in the molecular structure, as the material of the presentinvention. Here too, any of the aforesaid structures can be used asstructures adjoining the urea bonds, thiourea bonds or amide bonds, butit is most preferred that there be used both an aromatic compound and agroup (or groups) capable of forming a hydrogen bond, such as a compoundwhich possess hydroxyl, amino or carboxyl groups (including glucides orderivatives thereof).

Moreover, the material of the present invention may also be eithermonomer, oligomer or polymer, so material where an aforesaid structureor part thereof has been polymerized is also included in the materialsof the present invention. Thus, as the aforesaid structure or partthereof, there can be suitably employed synthetic polymers such asnylon, polymethyl methacrylate, polysulphone, polystyrene, polyethylene,polyvinyl alcohol, polytetrafluoroethylene and the like, or naturalpolymers including cellulose, collagen, chitin, chitosan and derivativesof these, etc. That is to say, there is ideally carried out theintroduction of groups capable of hydrogen bond formation intohomopolymer, copolymer or blended such synthetic polymers or naturalpolymers. Furthermore, there can also be appropriately used an inorganicmaterial such as metal, ceramic or glass which has been covered by asuitable polymer. The term carrier in the present invention denotes asupport on which the material of the present invention is fixed, and itmay be for example a synthetic polymer or natural polymer as exemplifiedabove.

The material of the present invention can be synthesized bygenerally-known methods. For example, in the case where a urea bond orthiourea bond is introduced into an aliphatic compound or an aromaticcompound, there can be used the method of performing reaction between anamino compound and an isocyanate compound or isothiocyanate compound.Furthermore, in the case of the introduction of an amide group into analiphatic compound or aromatic compound, there can be used for examplethe method of performing reaction between an acid, acid chloride or acidanhydride and an amino compound. Any mixing ratio of amino compound andisocyanate compound, isothiocyanate compound, acid, acid chloride oracid anhydride can be selected but, normally, there is desirably used0.1 to 10 mol of the amino compound per 1 mol of the isocyanatecompound, isothiocyanate compound, acid, acid chloride or acidanhydride. As the isocyanate compound or isothiocyanate compound, therecan be used any aliphatic isocyanate compound or isothiocyanate compoundsuch as, for example, ethyl isocyanate, stearyl isocyanate, n-butylisocyanate, iso-butyl isocyanate, n-propyl isocyanate, methylisothiocyanate, ethyl isothiocyanate, n-butyl isothiocyanate, benzylisothiocyanate, hexamethylene diisocyanate, cyclohexyl isocyanate,cyclohexyl isothiocyanate, cyclohexyl isothiocyanate, cyclohexyldiisocyanate and the like, but more preferably there is used an aromaticisocyanate compound or isothiocyanate compound such as phenylisocyanate, chlorophenyl isocyanate, fluorophenyl isocyanate,bromophenyl isocyanate, nitrophenyl isocyanate, tolyl isocyanate,methoxyphenyl isocyanate, 1-naphthyl isocyanate, 4,4′-diphenylmethanediisocyanate, 3,3,5,5′-tetraethyl-4,4′-diisocyanatodiphenylmethane,phenyl isothiocyanate, chlorophenyl isothiocyanate, fluorophenylisothiocyanate, nitrophenyl isothiocyanate, tolyl isothiocyanate,methoxyphenyl isothiocyanate, 1-naphthyl isothiocyanate and the like. Asthe acid chloride, there can be used any aliphatic acid chloride suchas, for example, isovaleryl chloride, stearoyl chloride,cyclohexanecarbonylchloride, 6-chloronicotinyl chloride and the likebut, more preferably, there can be used aromatic acid chlorides such asbenzoyl chloride, 3,4-dichlorobenzoyl chloride, nitrobenzoyl chloride,4-chlorobenzoyl chloride, 4-toluoyl chloride,benzo-[b]thiophene-2-carbonyl chloride and the like. Again, as the acidanhydride, there can desirably be used acetic anhydride, succinicanhydride, phthalic anhydride, benzoic anhydride and the like.Furthermore, the amino group in the amino compounds used in the presentinvention can be a primary amino group, secondary amino group ortertiary amino group and, as the amino compound, there can desirably beused, for example, ammonia, sec-octylamine, 1-(3-aminopropyl)imidazole,3-amino-1-propene, aminopyridine, aminobenzenesulphonic acid,tris(2-aminoethyl)amine and the like. Again, there can also be usedadvantageously a polyamino compound or an amino compound which possessesa hydroxyl group or carboxyl group, such that it is possible tointroduce a group or groups capable of forming a hydrogen bond inaddition to the urea, thiourea or amide bonds. As the polyaminocompound, there can be used any of, for example, diaminoethane,diethylenetriamine, triethylenetetramine, tetraethylenepentamine,dipropylenetriamine, N′-methyl-2,2′-diaminodiethylamine,polyethyleneimine, N-acetyl-ethylenediamine,1,2-bis(2-aminoethoxy)ethane and the like. As an amino compound with ahydroxy group, there can be used 2-ethanolamine, 3-propanolamine,6-hexanolamine, 1,3-diamino-2-hydroxypropane, 2-(2-aminoethoxy)ethanol,2-(2-aminoethylamino)ethanol, glucamine, N-methyl-1,3-diaminopropanol orother such aliphatic amine, or 4-aminophenol, diaminophenol,aminohydroxypyrimidine, diaminohydroxypyrimidine, diaminohydroxypyrazoleor other such aromatic amine, or serine, tyrosine or other such aminoacid. Again, by the reaction of epichlorohydrin and an amino compound,or 1,3-dibromo-2-hydroxypropane with a compound having only an aminogroup it is possible to synthesize an amino compound with a hydroxylgroup from a compound having only a hydroxyl group, or a compound onlyhaving an amino group. Furthermore, it is possible to use the samemethods as above in the case where a group capable of forming a hydrogenbond is introduced into a glucide. That is to say, in the case of aglucide with an amino group or amino groups such as chitosan orglucosamine, reaction can be carried out as described above withisocyanate compounds, isothiocyanate compounds, acids, acid chlorides oracid anhydrides. In the case of a glucide which does not possess aminogroups, amino groups can be introduced by activating hydroxyl groups inthe glucide with epichlorohydrin or tresyl chloride, and then reactingwith ammonia, diaminoethane or the like. By utilizing such amino groups,it is then possible to introduce groups capable of forming hydrogenbonds such as urea bonds, thiourea bonds, amide bonds or the like intothe glucide. As amino compounds with a carboxyl group, there can be usedfor example β-alanine, 4-amino-n-butyric acid,γ-amino-β-hydroxy-n-butyric acid, 6-amino-n-caproic acid or the like.

Furthermore, in the case where the material of the present invention isan oligomer or polymer, there is preferably used the method ofperforming reaction between the amino group of a compound with a groupwhich possesses a hydrogen bond forming capability with oligomer orpolymer which has isocyanate groups, carboxyl groups or carboxylic acidactive ester groups such as succinimide groups. Reacting an aforesaidisocyanate compound, isothiocyanate compound, acid, acid chloride oracid anhydride with an amino group-possessing oligomer or polymer, oroligomer or polymer in which amino groups have been introduced usingammonia, diaminoethane, 1,3-diaminopropane,1,3-diamino-2-hydroxypropane, 1,2-bis(2-aminoethoxy)ethane,tris(2-aminoethyl)amine, 2-(2-aminoethylamino)ethanol or the like, isalso a preferred method. Furthermore, controlling the reaction time,reaction temperature or mixing ratio, etc, or using protective groups,so that the acid chloride or isocyanate compound does not react with agroup or groups capable of forming a hydrogen bond, other than the aminogroup or groups, is desirable. Where required, functional groups such asamino groups, isocyanate groups, carboxyl groups or carboxylic acidactive ester groups such as succinimide groups can be introduced intothe oligomer or polymer.

Moreover, in the case where the material of the present invention is apolyurea or polythiourea, it is possible to use the method of performingreaction between, for example, a polyisocyanate compound orpolyisothiocyanate compound and a polyamino compound. Normally, in termsof the amount of reagents, there is desirably used from 0.1 to 10 mol ofthe polyamine per 1 mol of the polyisocyanate compound orpolyisothiocyanate compound. As the polyisocyanate compound orpolyisothiocyanate compound there is ideally employedhexamethylene-diisocyanate, cyclohexyldiisocyanate, tolylenediisocyanate, 4,4′-diphenylmethanediisocyanate,3,3′,5,5′-tetraethyl-4,4′-diisocyanatodiphenylmethane,xylene-diisocyanate, methylenebis(4-phenylisothiocyanate) or the like.Again, as the polyamino compound, there can be desirably employeddiaminoethane, diaminopropane, 1,3-diamino-2-hydroxypropane,N′-methyl-1,3-diamino-2-propanol, diaminophenol, N,N′-diaminopiperazine,diethylenetriamine, triethylenetetramine, tetraethylene-pentamine,polyethyleneimine, dipropylenetriamine,N′-methyl-2,2′-diaminodiethylamine or the like. Furthermore, in the casewhere the material of the present invention is a polyamide, there can beused for example the method of polycondensation of a polycarboxylic acidand a polyamine. Again, in the case of polyureas, polythioureas andpolyamides, there is also advantageously carried out the method where nopolyisocyanate, polyisothiocyanate, polycarboxylic acid, or the like, isused, and each functional group is introduced, one at a time, in turn,to finally obtain the polyurea, polythiourea or polyamide.

Again, the introduction of the hydrophobic group or groups can becarried out by known methods. Thus, preparation can readily be performedby carrying out reaction between a material with an amino group and thehalide of the hydrocarbon compound which constitutes the hydrophobicgroup, for example 1-bromobutane, 1-bromohexane or the like, or byfirstly performing activation of the hydroxyl groups of a hydroxyl groupcompound using epichlorohydrin, tresyl chloride or the like, after whichreaction is carried out with the amino derivative of a hydrocarboncompound, for example 1-aminobutane, 1-aminohexane or the like.

Moreover, ether bonds can be introduced by the reaction betweenbis(2-amino)ethoxyethane, polyoxyethylenebisamine or an amino sugar suchas glucosamine and activated ester groups or haloalkyl groups in thematerial.

All the above reactions are carried out, as a rule, at a reactiontemperature of 0 to 150° C. and a reaction time of 0.1 to 24 hours.Furthermore, while it is not absolutely necessary to employ a reactionsolvent, in general the reaction is carried out in the presence ofsolvent. As examples of the solvent, there are methanol, ethanol,isopropyl alcohol, n-butanol, hexane, acetone, N,N-dimethylformamide,dimethylsulphoxide and other such aliphatic hydrocarbons, benzene,toluene, xylene and other such aromatic hydrocarbons, dichloromethane,chloroform, chlorobenzene and other such halogenated hydrocarbons,diethyl ether, tetrahydrofuran, dioxane and other such ethers. Whererequired, following the end of the reaction, the reaction liquid issubjected to the usual post-treatment such as filtering andconcentration, after which it can be purified by column chromatography,recrystallization or other such process. Furthermore, in the case of awater-insoluble material, washing using a glass filter or the like isalso a preferred method.

Amongst the materials of the present invention, those that are waterinsoluble are favourably employed as, for example, a dressing or acolumn for removing LTA and/or PrA and/or aHL and/or proteinase. Theform thereof is not particularly restricted but, in the case where usedas a column, a form such as beads, fibre, hollow fibre, fibre bundles,yarn, net, knitted material, woven fabric or the like is preferred. Inthe case of a dressing, a woven material or film form is preferred.Furthermore, the material need not just be used on its own, but it canalso be used as one column or dressing, immobilized on a suitablematerial or mixed with some other material. The immobilizing or mixingstage, etc, may be carried out prior to, or after, processing in theaforesaid form. In the case where a column employing the material of thepresent invention is used as a column for extracorporeal circulation,the blood led out from the body may be directly passed through thecolumn or this may be used in combination with a plasma separationmembrane or the like.

Below, explanation is provided in further detail using examples, but thecontent of the invention is not to be restricted to the examples.

DESCRIPTION OF THE PREFERRED EMBODIMENTS EXAMPLE 1 PREPARATION OFMODIFIED POLYSTYRENE FIBRE

Islands-in-a-sea type composite fibre (thickness: 2.6 denier, number ofislands: 16) as described in U.S. Pat No. 4,661,260 (Example 1)comprising 50 weight ratio sea component (mixture of 46 weight ratio ofpolystyrene and 4 weight ratio of polypropylene) and 50 weight ratioislands component (polypropylene) was reacted for 1 hour at 20° C. witha solution mixture of 50 g of N-methylol-α-chloroacetamide, 400 g ofnitrobenzene, 400 g of 98% sulphuric acid and 0.85 g ofparaformaldehyde. Then, the fibre was washed with nitrobenzene andintroduced into water, to halt the reaction. Thereafter, by furtherwashing the fibre with hot water, there was obtainedchloroacetamidomethylated crosslinked polystyrene (hereinafter referredto as AMPSt).

The reagent shown in Table 1 was dissolved in 50 ml ofN,N-dimethylformamide (hereinafter abbreviated to DMF). To the solutionobtained, there was added 1 g of the AMPSt fibre (equivalent to a chlorocontent of 2 mmol) while stirring. Reaction was carried out at 30° C.for 3 hours. Subsequently, washing was conducted on a glass filter using200 ml of DMF and 200 ml of distilled water.

In this way, there were respectively produced materials with aminogroups (a)(b), amino groups and ether groups (c), amino groups andhydroxyl groups (d), and amino groups and hydrophobic groups (e) to (h),introduced into a carrier fibre having amide bonds.

In addition, prior to washing aforesaid materials (a) to (h) withdistilled water, they were also respectively added to a solutioncomprising 0.19 g of 4-chlorophenylisocyanate dissolved in 50 ml of DMF,and reaction was carried out for 1 hour at 25° C. Thereafter, washingwas carried out on a glass filter using 200 ml of DMF and 200 ml ofdistilled water, and the amino groups converted to urea derivatives.(a2) to (h2)

TABLE 1 Reagents used for Reaction with AMPst Fibre Reaction ReagentEmployed in the Amount of Product Reaction Reagent (g) (a)tetraethylenepentamine 0.3 (b) ethylenediamine 0.3 (c)1,2-bis(2-aminoethoxy)ethane 0.8 (d) 1,3-diamino-2-hydroxypropane 0.5(e) n-butylamine 0.8 (f) n-hexylamine 0.8 (g) n-octylamine 0.8 (h)n-hexadecylamine 0.8

EXAMPLE 2 Introduction of Urea Groups, Thiourea Groups and Amide Groupsinto Chitosan Beads

12 ml (volume when precipitated; dry weight 1.0 g) of chitosan beads ofdiameter 0.1 mm (produced by Fujibo (Ltd), “Chitopearl”AL-01) werestirred in 50 ml of DMF, and then the beads and solvent separated bymeans of a glass filter. This procedure was carried out 20 times, for 5minutes per time, and the water component content completely replaced byDMF.

The beads were then slowly added to a solution of the reagent shown inTable 2 dissolved in 100 ml of DMF and reaction carried out for 1 hourat room temperature, while stirring. Subsequently, using a glass filter,the beads and solution were separated and the beads washed by stirringfor 5 minutes in 50 ml of DMF. This washing procedure was repeated 20times, and unreacted reagent completely removed. Next, the washingprocedure was carried out in the same way by means of distilled water,to replace the DMF with distilled water and, in this way, chitosan beadscontaining urea bonds, thiourea bonds or amide bonds were obtained.

TABLE 2 Reaction Amount of Product Reagent Employed in the ReactionReagent (g) (i) 4,4′-diphenylmethanediisocyanate 0.7 (j) 4-chlorophenylisothiocyanate 0.5 (k) 4-chlorobenzoyl chloride 0.5

EXAMPLE 3 Introduction of Amino Groups and Hydrophobic

Groups into Cellulose Beads

Water was added to 200 ml of cellulose beads of diameter 0.1 mm(produced by the Chisso Corporation, “Cellofine GCL-700 m”) until thevolume was 400 ml, after which 40 ml of 5N aqueous sodium hydroxidesolution was added, then 50 ml of epichlorohydrin added and reactioncarried out for 6 hours at 40° C. After the reaction, the beads werewashed with 3000 ml of distilled water on a glass filter and there wasobtained cellulose beads with epoxy groups introduced (EPO).

10 ml of the EPO beads was slowly added to a solution of the reagentshown in Table 3 dissolved in 100 ml of a 50% (v/v) aqueous ethanolsolution and, while stirring, reaction was carried out for 7 days at 50°C. Subsequently, using a glass filter, the beads and solution wereseparated, and the beads washed with 500 ml of 50% (v/v) aqueous ethanolsolution and then with 500 ml of distilled water, to completely removeunreacted reagent. In this way, amino groups and hydrophobic groups wereintroduced into the cellulose beads to give the following structure.

 X¹=OH and X²=NH.

Y = (CH₂)₃CH₃ (l) = (CH₂)₅CH₃ (m) = (CH₂)₇CH₃ (n)

n=1

TABLE 3 Reaction Reagent Used in Amount of Product the Reaction log PReagent (g) (l) n-butylamine 0.77 0.7 (m) n-hexylamine 1.93 0.5 (n)n-octylamine 2.90 0.5

EXAMPLE 4 Test to Remove LTA, PrA, aHL, Proteinase, Endotoxin andSuperantigen by Means of the Modified AMPSt Fibre Modified ChitosanBeads and Modified Cellulose

A test was carried out of the adsorption and removal of LTA, PrA, aHL,proteinase, endotoxin and superantigen by means of the modified AMPStfibre prepared in Example 1, the modified chitosan beads prepared inExample 2 and the modified cellulose beads prepared in Example 3. TheLTA was derived from Staphylococcus aureus (obtained from the SigmaCo.); the PrA was obtained from the Prozyme Co.; the endotoxin(hereinafter abbreviated to Et) was standard product derived from E.coli strain 0111B4 (obtained from the Difco Co.); and the proteinase,namely SPEB, and the aHL and TSST-1 were obtained from the ToxinTechnology Co. The removal test was conducted using rabbit plasma. Bloodwas taken from rabbit using an anticoagulant, and the plasma wasobtained by centrifuging for 15 minutes at 3,000 rpm. The plasma washeated for 2 hours at 56° C. and heat-inactivated plasma prepared. Thisplasma was filtered with a 0.45 μm fine diameter membrane filter, afterwhich LTA, PrA, aHL, SPEB, Et and TSST-1 were added at concentrations of10 μg/ml respectively. 35 mg of the modified AMPSt fibre of Table 1, 0.1ml (precipitated volume) of the modified chitosan beads of Table 2 or0.1 ml (precipitated volume) of the modified cellulose beads wererespectively added to 0.5 ml of the LTA-added serum, and shakingperformed for 2 hours at 37° C. The concentrations of thebacterially-derived components in the solution after 2 hours weremeasured by an enzyme immunological measurement method (LTA, PrA, aHL,SPEB, TSST-1) or by using Limulus reagent (obtained from Wako PureChemical Industries). The results are shown in Table 4. These resultsindicate that, unlike a material such as polystyrene which does notpossess groups capable of forming hydrogen bonds, the materials with ahydrogen bond forming capability such as urea bonds, thiourea bonds,amide bonds, amino groups or hydroxyl groups remove, at the same time,from one to five of LTA, PrA, aHL, SPEB, Et and TSST-1. Furthermore,hydrophobic groups and ether bonds show an effect in promoting theremoval ability. Again, it is apparent that, where the log P value ofthe hydrophobic group is at least 0.7, it is possible to add a bondingcapacity in terms of LTA, PrA, aHL, SPEB, Et or TSST-1.

Now, Table 4 shows the percentage removal Ar calculated, from theconcentration (C₀) of the toxin prior to the removal test and theconcentration (C) after removal, by means of formula 1.

Ar=(C₀−C)/C₀  Formula 1

TABLE 4 Test to remove LTA, PrA, aHL, proteinase, endotoxin orsuperantigen by means of the modified AMPSt fibre and modified chitosanLTA PrA aHL SPEB Et TSST-1 (%) (%) (%) (%) (%) (%) unmodified fibre 0 00 0 0 0 (a) 85 25 32 47 70 48 (b) 53 33 18 43 53 40 (c) 48 23 18 39 4237 (d) 41 17 20 51 38 53 (e) 12 20 18 38 20 42 (f) 50 14 12 39 49 35 (g)82 10 9 32 60 28 (h) 78 21 13 30 59 46 (a2) 84 62 78 93 62 86 (b2) 65 5674 89 40 81 (c2) 62 52 79 78 25 80 (d2) 67 61 39 96 19 82 (e2) 13 24 2175 14 76 (f2) 25 38 24 78 17 63 (g2) 42 46 38 67 32 49 (h2) 58 51 41 5830 58 unmod. chitosan 5 2 8 3 27 6 (i) 95 53 72 96 54 89 (j) 58 62 62 8548 80 (k) 52 45 52 71 42 69 unmod. PS beads 0 0 0 0 0 0 unmod. cellbeads 6 2 0 6 0 4 (l) 31 36 11 23 10 12 (m) 55 50 34 37 15 33 (n) 80 4277 90 21 84 unmodified fibre = unmodified polystyrene composite fibreunmod. chitosan = unmodified chitosan beads unmod. PS beads = unmodifiedpolystyrene beads unmod. cell beads = unmodified cellulose beads

EXAMPLE 5 LTA PrA, aHL, SPEB, Et, TSST-1; Removal Test by Means ofModified AMPSt Fibre (circulatory method)

LTA, PrA, aHL, SPEB, Et and TSST-1 removal tests were conducted usingthe modified AMPSt fibre (a2) of Example 1, by means of a circulatorymethod. 1 g of fibre was introduced into a column in each case, andTable 5 shows the LTA concentration when 10 ml of the rabbit plasmaprepared by the method in Example 3 was circulated through the column at37° C. for 60 minutes at rate of 1 ml/min. Even under extracorporealcirculation type flow conditions, the concentration fell with passage oftime and a removal capability was shown.

In the same way as in Example 4, the percentage removal Ar calculatedfrom the concentration of each toxin before the removal test (C₀) andthe concentration (C) after removal over a given time, by means offormula 1, is shown in the table.

TABLE 5 LTA PrA aHL SPEB Et TSST-1 % % % % % % percentage removal priorto 0 0 0 0 0 0 circulation percentage removal after 15 65 56 74 78 40 67minutes percentage removal after 30 62 52 79 78 51 79 minutes percentageremoval after 60 82 57 70 91 62 82 minutes

EXAMPLE 6 Preparation of an Adsorbent Body and the Measurement of theRemoval of Bacterially-derived Components and an Antibiotic Using thisAdsorbent Body

Using the α-chloroacetamidomethylated crosslinked polystyrene fibreprepared in Example 1 (hereinafter referred to as AMPSt), 20 g of thisAMPst fibre was added, while stirring, to a liquid comprising 6.3 g oftetraethylenepentamine and 7.2 g of n-butylamine dissolved in 500 ml ofDMF. Reaction was carried out for 3 hours at 30° C., after which thereacted fibre was washed on a glass filter using 500 ml of DMF.Furthermore, this fibre was added to a solution of 2.3 g of4-chlorophenylisocyanate dissolved in 500 ml of DMF, and reactioncarried out for 1 hour at 25° C. Thereafter, washing was carried out onthe glass filter using 1000 ml of DMF and also using 1000 ml ofdistilled water, and AMPst fibre containing introduced urea bonds wasobtained (hereinafter referred to as UAMP fibre).

The removal of bacterially-derived components and an antibiotic usingthe prepared UAMP fibre was tested. As bacterially-derived components,an adsorption removal test was carried for PrA, aHL, proteinase,endotoxin and superantigen. As the antibiotic there was employedArbekacin (Meiji Seika Kaisha Ltd., hereinafter abbreviated to as ARK)and as a pressor there was used adrenaline (made by Daiichi Seiyaku).The concentration was measured enzyme-immunologically in the case of theTSST-1, α-hemolysin, protein A and SPEB; by means of the Limulus reagent(Wako Pure Chemical Industries).in the case of the Et; by means of afluorescence polarization immunoassay method in the case of ARK; and byliquid chromatography in the case of the adrenaline.

The liquid subjected to component removal was produced by dissolving 1μg/ml of PrA, aHL, TSST-1 and SPEB, 10 μg/ml of LPS, 10 μg/ml of ARK and100 μg/ml of adrenaline, in 0.1 M sodium phosphate buffer (pH 7.4)containing 0.15 m/l sodium chloride and 5 mg/ml of bovine serum albumin(Fraction V)(produced by the Seikagaku Kogyo Co.). 0.1 g of the UAMP wasadded to the test liquid and the removal reaction carried out whileconducting rotary stirring for 2 hours at 37° C. The concentrations weremeasured before and after the removal in each case and the percentageremoval calculated using aforesaid formula (1). The results are shown inTable 6.

TABLE 6 Test of the Removal of Bacterially-Derived Components and anAntibiotic Material Concentration Concentration Undergoing C₀ Prior toafter Removal Percentage Removal Removal ng/ml ng/ml Removal α-hemolysin1 0.38 62 protein A 1 0.46 54 SPEB 1 0.13 87 TSST-1 1 0.20 80 LPS 10 4.258 ARK 0.01 0.0093 7 adrenaline 100 89 11

Effects of the Invention

By means of the present invention, there is provided a materialcontaining functional groups capable of forming hydrogen bonds, whichenables LTA and/or PrA and/or aHL and/or proteinase to be rapidlydetoxified or removed in solutions of high protein concentration such asblood plasma. Since it is possible, using the material of the presentinvention, to bring about loss of toxic activity (detoxification) of LTAand/or PrA and/or aHL and/or proteinase present in pharmaceuticalproducts or in body fluids such as blood and plasma, there becomespossible the treatment or prevention of septic shock, infectious diseaseand the like. Furthermore, where water-insoluble materials are employedfor this material, it is possible to efficiently remove LTA and/or PrAand/or aHL and/or proteinase from within body fluids such as blood orplasma, or drugs, so by producing a column or dressing it becomespossible therewith to treat or prevent septic shock, infections or thelike.

What is claimed is:
 1. A bacterially-derived component detoxification orremoval material having one or more than one functional group capable ofhydrogen bond formation and detoxifies or removes at least one of thebacterially-derived components selected from lipoteichoic acid, proteinA, α hemolysin and proteinase.
 2. A bacterially-derived componentdetoxification or removal material according to claim 1 where saidbacterially-derived component is lipoteichoic acid.
 3. A method for thedetoxification or removal of lipoteichoic acid from liquid by passingliquid containing lipoteichoic acid through a column packed withmaterial according to claim
 2. 4. A bacterially-derived componentdetoxification or removal material according to claim 1 where saidbacterially-derived component is protein A.
 5. A method for thedetoxification or removal of protein A from liquid by passing liquidcontaining protein A through a column packed with material according toclaim
 4. 6. A method for the detoxification or removal of proteinasefrom liquid by passing liquid containing proteinase through a columnpacked with material according to claim
 4. 7. A bacterially-derivedcomponent detoxification or removal material according to claim 1 wheresaid bacterially-derived component is α hemolysin.
 8. A method for thedetoxification or removal of α hemolysin from liquid by passing liquidcontaining α hemolysin through a column packed with material accordingto claim
 7. 9. A bacterially-derived component detoxification or removalmaterial according to claim 1 where said bacterially-derived componentis proteinase.
 10. A method according to any one of claims 4, 7 or 9wherein the liquid is blood or plasma.
 11. A detoxification or removalmaterial according to claim 1 where the bacterially-derived componentcomprises at least two bacterially derived components selected fromlipoteichoic acid, protein A, α hemolysin and proteinase.
 12. A methodwhere, by passing liquid containing two or more components selected fromlipoteichoic acid, protein A, α hemolysin and proteinase, through acolumn packed with material according to claim 11, two or more of saidcomponents are detoxified or removed from the liquid.
 13. A materialaccording to claim 1 wherein the one or more than one functional groupcapable of hydrogen bond formation is selected from the urea bond,thiourea bond, amide bond, amino group and hydroxyl group.
 14. Amaterial according to claim 1 having two or more of said functionalgroups capable of hydrogen bond formation.
 15. A material according toclaim 14 having functional groups of different chemical structurecapable of hydrogen bond formation.
 16. A material according to claim 14wherein at least one of the groups capable of hydrogen bond formation isan amino group.
 17. A material according to claim 16 wherein the aminogroup is secondary or tertiary.
 18. A material according to claim 16wherein the amino group is a polyamino group.
 19. A material accordingto claim 14 wherein at least one of the groups capable of hydrogen bondformation is a hydroxyl group.
 20. A material according to claim 19wherein the hydroxyl group is glucide-derived.
 21. A material accordingto claim 20 wherein the glucide is selected from chitosan, cellulose andderivatives thereof.
 22. A material according to claim 1 having at leastone hydrophobic group.
 23. A material according to claim 22 wherein thehydrophobic group is a hydrocarbon chain with four or more carbons. 24.A material according to claim 22 wherein the hydrophobic group is anaromatic ring.
 25. A material according to claim 22 wherein when thepartition coefficient for an octanol/water system is taken as P, log Pof the hydrophobic group is 0.5 or more.
 26. A material according toclaim 22 having two or more types of hydrophobic group.
 27. A materialaccording to claim 22 wherein the distance from the group capable ofhydrogen bond formation to the hydrophobic group is no more than fiveatoms.
 28. A material according to claim 1 having an ether bond.
 29. Amaterial according to claim 28 wherein the ether bond isglucide-derived.
 30. A material according to claim 29 wherein theglucide is selected from chitosan, cellulose and derivatives thereof.31. A material according to claim 1 which comprises a group representedby the following formula:

X: functional group capable of hydrogen bond formation, where X¹ and x²may be the same or different Y: an alkyl group with four or more carbonsor an aromatic ring n: 0-5.
 32. A material according to claim 31 where Xis selected from an amino group and a hydroxyl group.
 33. A materialaccording to claim 1 including a carrier.
 34. A material according toclaim 33 wherein the carrier is selected from polystyrene, polysulfone,polymethyl methacrylate, cellulose and chitosan, and derivativesthereof.
 35. A material according to claim 33 wherein the carrier is afiber.
 36. A material according to claim 35 the fiber is anislands-in-a-sea fiber.
 37. A material according to claim 1 which iswater-insoluble.
 38. A material according to claim 1 for use in sepsistreatment.
 39. A body fluid purification column which comprises amaterial according to claim
 1. 40. A dressing which comprising materialaccording to claim 1.